Microscopic and Biopharmaceutical Evaluation of Emulsion and Self-Emulsifying Oil with Cyclosporine.

Among the currently available commercial eye drops with cyclosporine A (Cs) there is a lack of long-acting dosage forms and products with a concentration of the drug substance higher than 0.1%, although Cs is widely used in ophthalmology. The aim of the research was to conduct the microscopic and biopharmaceutical evaluation of two formulations, an emulsion (EM) and a self-emulsifying oil (SEO), both with 0.5% of Cs, proposed for use in eye drops, and the comparison of both. SEO eye drops with Cs or any other drug substance are currently not available as marketed products, and the highest concentration of Cs in the ocular emulsion is only 0.1%. The microscopic evaluation of the emulsion and the SEO after emulsification with water was carried out using a high-resolution digital microscopy. The properties of both preparations were compared using the high dynamic range function or optical shadow effect mode. Images in the 3D composition mode were also recorded. The in vivo study of the Cs formulations was performed on male albino rabbits. The eye tolerance of the preparations was assessed using the ocular irritation test, which is a modified Draize test. Placebo carriers (without the drug substance) were also subjected to irritation testing. The concentration of Cs in the tissues (cornea and conjunctiva) and fluids (tear fluid and aqueous humor) of the rabbit eye was determined after multiple instillations of Cs-EM or Cs-SEO. The tested preparations were compared using the digital microscopy technique, which highlights the features of the formulations and eliminates the risk of unnoticeable properties that are difficult to observe in classical optical microscopy. Both tested Cs-loaded formulations are classified as practically non-irritating. There were also no significant differences when testing the placebo carriers. After a topical administration, Cs was widely distributed in all tissues (e.g., in cornea 1.3 ng/mg and 1.0 ng/mg) and fluids of the eye (e.g., in tear fluid 11.6 µg/mL and 4.3 µg/mL), after the administration of Cs-SEO and Cs-EM, respectively. The obtained results allow us to recognize both tested formulations, the emulsion and the self-emulsifying oil with 0.5% Cs content, as carriers safe for ophthalmic use and effective in delivering the drug substance to the structures of the eye.

Reversed-phase pH gradient thin-layer chromatography of biologically active substances with controlled developing solvent velocity.

For the first time, stepwise pH gradient thin-layer chromatograms of biologically active substances with controlled developing solvent velocity are presented and described in the paper. Change in buffer pH of the mobile phase solution influences retardation, selectivity, and shape of the separated substances' spots. The conducted research has confirmed that the mobile phase's pH gradient could be an essential factor to optimize the conditions of the separation of substances in reversed-phase high-performance thin-layer chromatography. The reproducibility of the gradient retardation factor values of separated substance zones is satisfactory.

The Characterization of Ground Raspberry Seeds and the Physiological Response to Supplementation in Hypertensive and Normotensive Rats.

This study aimed to evaluate the protective role of ground raspberry seeds (RBS) as a source of polyphenols and essential fatty acids on blood plasma enzymatic antioxidant status, lipid profile, and endothelium-intact vasodilation during physiological and pathological conditions. Young normotensive Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) at ten weeks of age were fed with either a control diet or were supplemented with added 7% RBS for six weeks (n = 6). The main component of RBS was dietary fiber (64%) and the main polyphenols were ellagitannins (1.2%) and flavan-3-ols (0.45%). Irrespective of the rat model, ground RBS decreased liver enzyme aspartate aminotransferase (0.9-fold) and hydrogen peroxide scavenging capacity (Catalase, 0.9-fold). In supplemented SHRs, preincubation with inducible nitric oxide synthase (iNOS) inhibitor 1400W, nonselective cyclooxygenase (COX) inhibitor indomethacin, selective COX-2 inhibitor NS-398, prostacyclin (PGI2) synthesis inhibitor tranylcypromine (TCP), thromboxane receptor (TP) antagonist SQ-29548, thromboxane synthesis inhibitor furegrelate, and 20-HETE synthesis inhibitor HET0016 induced the same relaxant response to acetylcholine as in the nonsupplemented control group. In supplemented WKYs, atherogenic index was decreased (0.8-fold), while iNOS and COX-2-derived PGI2 increased acetylcholine-induced vasodilation. These effects of ground RBS may constitute a potential mechanism for preventing cardiovascular diseases.

Metabolomic Signature of Early Vascular Aging (EVA) in Hypertension.

Arterial stiffening is a hallmark of early vascular aging (EVA) syndrome and an independent predictor of cardiovascular morbidity and mortality. In this case-control study we sought to identify plasma metabolites associated with EVA syndrome in the setting of hypertension. An untargeted metabolomic approach was used to identify plasma metabolites in an age-, BMI-, and sex-matched groups of EVA (n = 79) and non-EVA (n = 73) individuals with hypertension. After raw data processing and filtration, 497 putative compounds were characterized, out of which 4 were identified as lysophosphaditylcholines (LPCs) [LPC (18:2), LPC (16:0), LPC (18:0), and LPC (18:1)]. A main finding of this study shows that identified LPCs were independently associated with EVA status. Although LPCs have been shown previously to be positively associated with inflammation and atherosclerosis, we observed that hypertensive individuals characterized by 4 down-regulated LPCs had 3.8 times higher risk of EVA compared to those with higher LPC levels (OR = 3.8, 95% CI 1.7-8.5, P < 0.001). Our results provide new insights into a metabolomic phenotype of vascular aging and warrants further investigation of negative association of LPCs with EVA status. This study suggests that LPCs are potential candidates to be considered for further evaluation and validation as predictors of EVA in patients with hypertension.

Bayesian multilevel model of micro RNA levels in ovarian-cancer and healthy subjects.

In transcriptomics, micro RNAs (miRNAs) has gained much interest especially as potential disease indicators. However, apart from holding a great promise related to their clinical application, a lot of inconsistent results have been published. Our aim was to compare the miRNA expression levels in ovarian cancer and healthy subjects using the Bayesian multilevel model and to assess their potential usefulness in diagnosis. We have analyzed a case-control observational data on expression profiling of 49 preselected miRNA-based ovarian cancer indicators in 119 controls and 59 patients. A Bayesian multilevel model was used to characterize the effect of disease on miRNA levels controlling for differences in age and body weight. The difference between the miRNA level and health status of the patient on the scale of the data variability were discussed in the context of their potential usefulness in diagnosis. Additionally, the cross-validated area under the ROC curve (AUC) was used to assess the expected out-of-sample discrimination index of a different sets of miRNAs. The proposed model allowed us to describe the set of miRNA levels in patients and controls. Three highly correlated miRNAs: miR-101-3p, miR-142-5p, miR-148a-3p rank the highest with almost identical effect sizes that ranges from 0.45 to 1.0. For those miRNAs the credible interval for AUC ranged from 0.63 to 0.67 indicating their limited discrimination potential. A little benefit in adding information from other miRNAs was observed. There were several miRNAs in the dataset (miR-604, hsa-miR-221-5p) for which inferences were uncertain. For those miRNAs more experimental effort is needed to fully assess their effect in the context of new hits discovery and usefulness as disease indicators. The proposed multilevel Bayesian model can be used to characterize the panel of miRNA profile and to assess the difference in expression levels between healthy and cancer individuals.

Urinary metabolomic signature of muscle-invasive bladder cancer: A multiplatform approach.

Bladder cancer (BCa) is ninth amongst the most common types of cancer in the human population worldwide. The statistics of incidence and mortality of BCa are alarming and the currently applied diagnostic methods are still not sensitive enough. This leads to a large number of undiagnosed BCa cases, usually among patients in the early stages of the disease. Despite the fact that many risk factors of BCa have been recognized, the pathomechanism of development of bladder cancer has not been fully explained yet. Therefore, in the present study, multiplatform urinary metabolomics has been implemented in order to scrutinize potential diagnostic indicators of BCa that might help to explain its pathomechanism and be potentially useful in diagnosis and determination of stage of the disease. Urine samples collected from muscle-invasive high grade BCa patients (n = 24) and healthy volunteers (n = 24) were matched in terms of most common BCa risk factors i.e. gender, age, BMI and smoking status. They were analyzed by high performance liquid chromatography coupled with time of flight mass spectrometry detection (HPLC-TOF/MS) using RP and HILIC chromatography, gas chromatography hyphenated with triple quadruple mass spectrometry detection (GC-QqQ/MS) in scan mode, and proton nuclear magnetic resonance (1H NMR). The six datasets obtained were submitted to univariate and multivariate statistical analyses. 17 metabolites significantly discriminated urinary profiles of BCa patients from urinary profiles of healthy volunteers. These metabolites are mainly involved in amino acid metabolism, pyrimidine and purine metabolism, as well as energy metabolism and might play a crucial role in the pathogenesis of BCa.

Column Characterization and Selection Systems in Reversed-Phase High-Performance Liquid Chromatography.

Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most popular chromatographic mode, accounting for more than 90% of all separations. HPLC itself owes its immense popularity to it being relatively simple and inexpensive, with the equipment being reliable and easy to operate. Due to extensive automation, it can be run virtually unattended with multiple samples at various separation conditions, even by relatively low-skilled personnel. Currently, there are >600 RP-HPLC columns available to end users for purchase, some of which exhibit very large differences in selectivity and production quality. Often, two similar RP-HPLC columns are not equally suitable for the requisite separation, and to date, there is no universal RP-HPLC column covering a variety of analytes. This forces analytical laboratories to keep a multitude of diverse columns. Therefore, column selection is a crucial segment of RP-HPLC method development, especially since sample complexity is constantly increasing. Rationally choosing an appropriate column is complicated. In addition to the differences in the primary intermolecular interactions with analytes of the dispersive (London) type, individual columns can also exhibit a unique character owing to specific polar, hydrogen bond, and electron pair donor-acceptor interactions. They can also vary depending on the type of packing, amount and type of residual silanols, "end-capping", bonding density of ligands, and pore size, among others. Consequently, the chromatographic performance of RP-HPLC systems is often considerably altered depending on the selected column. Although a wide spectrum of knowledge is available on this important subject, there is still a lack of a comprehensive review for an objective comparison and/or selection of chromatographic columns. We aim for this review to be a comprehensive, authoritative, critical, and easily readable monograph of the most relevant publications regarding column selection and characterization in RP-HPLC covering the past four decades. Future perspectives, which involve the integration of state-of-the-art molecular simulations (molecular dynamics or Monte Carlo) with minimal experiments, aimed at nearly "experiment-free" column selection methodology, are proposed.

Metabolomic Heterogeneity of Urogenital Tract Cancers Analyzed by Complementary Chromatographic Techniques Coupled with Mass Spectrometry.

BACKGROUND: In regard to urogenital tract cancer studies, an estimated 340,650 new cases and 58,360 deaths from genital system cancer and about 141,140 new cases and 29330 deaths from urinary system were projected to occur in the United States in 2012. The main drawbacks of currently available diagnostic tests constitute the low specificity, costliness and quite high invasiveness. OBJECTIVE: The main goal of this pilot study was to determine and compare urine metabolic fingerprints in urogenital tract cancer patients and healthy controls. METHOD: A comparative analysis of the metabolic profile of urine from 30 patients with cancer of the genitourinary system (bladder (n=10), kidney (n=10) and prostate (n=10)) and 30 healthy volunteers as a control group was provided by LC-TOF/MS and GCQqQ/ MS. The data analysis was performed by the use of U-Mann Whitney test or Student's t-test, principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). RESULTS: As a result, 33, 43, and 22 compounds were identified as statistically significant in bladder, prostate and kidney cancer, respectively, compared to healthy groups. CONCLUSION: Diverse compounds such as purine, sugars, amino acids, nucleosides, organic acids which play a role in purine metabolism, in tricarboxylic acid cycle, in amino acid metabolism or in gut microbiota metabolism were identified. Only two metabolites namely glucocaffeic acid and lactic acid were found to be in common in studied three types of cancer.

Untargeted Metabolomics Provides Insight into the Mechanisms Underlying Resistant Hypertension.

BACKGROUND: Resistant hypertension (RH) affects about 15-20% of treated hypertensive patients worldwide. RH increases the risk of cardiovascular events such as myocardial infarction and stroke by 50%. The pathological mechanisms underlying resistance to treatment are still poorly understood. OBJECTIVE: The main goal of this pilot study was to determine and compare plasma metabolomic profiles in resistant and non-resistant hypertensive patients. METHODS: We applied untargeted metabolomic profiling in plasma samples collected from 69 subjects with RH and 81 subjects with controlled hypertension. To confirm patients' compliance to antihypertensive treatment, levels of selected drugs and their metabolites were determined in plasma samples with the LC-ESI-TOF/MS technique. RESULTS: The results showed no statistically significant differences in the administration of antihypertensive drug in the compared groups. We identified 19 up-regulated and 13 downregulated metabolites in the RH. CONCLUSION: The metabolites altered in RH are linked to oxidative stress and inflammation, endothelium dysfunction, vasoconstriction and cell proliferation. Our results may generate new hypothesis about RH development and progression.

Targeted metabolomics in bladder cancer: From analytical methods development and validation towards application to clinical samples.

Bladder cancer constitutes the ninth most common cancer worldwide and, despite continuous development of new diagnostic approaches, the thirteenth leading cause of global cancer mortality. In our previous untargeted urine metabolomic investigation, seventeen metabolites were found to be statistically differentiating bladder cancer patients and healthy volunteers. Therefore, the main goal of this study was to develop and validate an analytical method for simultaneous quantitative determination of those metabolites using reversed phase high-performance liquid chromatography coupled with triple quadrupole mass spectrometry technique (RP-HPLC-QQQ/MS). Different chromatographic conditions, as well as various sample treatment procedures were tested in order to provide the best separation and the lowest limit of quantification (LOQ) values for studied compounds. The validation was performed according to the Food and Drug Administration guidelines (FDA). The limit of determination (LOD) and the LOQ values were in the range of 0.21-10.51 ng/ml and 0.69-35.02 ng/ml, respectively. The concentration range of compounds was developed between 2.5 and 12500 ng/ml. Only one compound (trimethyllysine) showed a significant matrix effect (61%) and consequently low process efficiency (64%). Overall, developed method presented recovery and precision values within the ranges proposed by FDA guidelines. The optimized and validated method was applied to urine samples obtained from 40 patients with bladder cancer and 40 healthy volunteers matched according to ones of the most important risk factors for developing urinary bladder tumors, e.i. age, gender and BMI. Afterwards, statistical analysis was provided by the use of Student's t-test or U-Mann Whitney test. The developed method was sensitive, selective and reproducible to be applied for the quantification of metabolites in the investigation of urine samples. As a consequence, ten out of previously chosen seventeen compounds, participating in different metabolites' pathways (gut floral metabolism, RNA degradation, purine metabolism, etc.), were found to be statistically significantly different in the urine concentration (p < 0.05) between cancer and control groups.

Analysis of Isocratic-Chromatographic-Retention Data using Bayesian Multilevel Modeling.

The objective of this work was to develop a multilevel (hierarchical) model based on isocratic-reversed-phase-high-performance-chromatographic data collected in methanol and acetonitrile for 58 chemical compounds. Such a multilevel model is a regression model of the analyte-specific chromatographic measurements, in which all the regression parameters are given a probability model. It is a fundamentally different approach from the most common approach, where parameters are separately estimated for each analyte (without sharing information across analytes and different organic modifiers). The statistical analysis was done with Stan software implementing the Bayesian-statistics inference with Markov-chain Monte Carlo sampling. During the model-building process, a series of multilevel models of different complexity were obtained, such as (1) a model with no pooling (separate models were fitted for each analyte), (2) a model with partial pooling (a common distribution was used for analyte-specific parameters), and (3) a model with partial pooling as well as a regression model relating analyte-specific parameters and analyte-specific properties (QSRR equations). All the models were compared with each other using 10-fold cross-validation. The benefits of multilevel models in inference and predictions were shown. In particular the obtained models allowed us to (i) better understand the data and (ii) solve many routine analytical problems, such as obtaining well-calibrated predictions of retention factors for an analyte in acetonitrile-containing mobile phases given zero, one, or several measurements in methanol-containing mobile phases and vice versa.

Ocular irritation and cyclosporine A distribution in the eye tissues after administration of Solid Lipid Microparticles in the rabbit model.

The aim of this study was to investigate the in vivo effect of Solid Lipid Microparticles (SLM), proposed for topical ocular administration of cyclosporine, on the rabbit eye. SLM carrier is an aqueous dispersion of lipid microparticles (20% w/w) with a size up to 15 µm. Cyclosporine was dissolved in the formulation in the concentration of 0.5 or 2.0% (w/w). Ocular tolerance of microsphere dispersion was assessed in rabbit model by the Draize eye test (SLM was compared with emulsion and oily solution), and cyclosporine distribution in ocular tissues was evaluated after multiple application of tested formulations (SLM dispersions, emulsions and oily solution) for 7 days. Good tolerance of cyclosporine-SLM formulation was demonstrated in the rabbit model. Concentration of cyclosporine in the precorneal tissues, such as cornea and conjunctiva, was much higher than the therapeutic value (8.4 ng/mg and 3.2 ng/mg, respectively). After SLM administration, the cyclosporine concentrations determined in the anterior ocular tissues, were also significantly higher compared to those obtained after the application of other tested carriers (emulsions and oily solution). The obtained results prove that the recognized SLM dispersions are safe formulations for ophthalmic use. It can be concluded that lipid microparticles are highly promising for an efficient ophthalmic drug delivery, when compared to other conventional dosage forms.

Free silanols and ionic liquids as their suppressors in liquid chromatography.

In this review, we will firstly discuss the types and the general properties of silica, focusing on the silica support used in chromatography and capillary electrophoresis. Additionally, the characterization of functional groups (silanols and siloxanes) will be considered in terms of activity of the stationary phases. We will then discuss physical chemistry of the stationary phases applied in liquid chromatography and capillary electrophoresis. The use of ionic liquids as a silanols' suppressors will be presented in the next parts of the study, along with the examples of specific applications. The review is completed with conclusions and an outlook for the future developments in the area of analytical applications of ionic liquids.

Quantitative determination of trigonelline in mouse serum by means of hydrophilic interaction liquid chromatography-MS/MS analysis: Application to a pharmacokinetic study.

Trigonelline is a pyridine alkaloid found in fenugreek seeds and coffee beans. Most of the previous studies are concerned with the quantification of trigonelline along with other constituents in coffee herbs or beverages. Only a few have focused on its determination in animal or human tissues by applying different modes of HPLC with UV or MS detection. The aim of the study was to develop and validate a fast and simple method for trigonelline determination in serum by the use of hydrophilic interaction liquid chromatography (HILIC) with ESI-MS/MS detection. Separation of trigonelline was achieved on a Kinetex HILIC column operated at 35°C with acetonitrile-ammonium formate (10 mm, pH = 3) buffer mixture (55:45, v/v) as the mobile phase. The developed method was successfully applied to determine trigonelline concentration in mouse serum after intravenous administration of 10 mg/kg. The developed assay is sensitive (limit of detection = 1.5 ng/mL, limit of quantification = 5.0 ng/mL) and linear in a concentration range from 5.0 to 250.0 ng/mL. Sample preparation is limited to deproteinization, centrifugation and filtration. The application of the HILIC mode of chromatography with MS detection and selection of deuterated trigonelline as internal standard allowed a rapid and precise method of trigonelline quantification to be to developed.

How to model temporal changes in nontargeted metabolomics study? A Bayesian multilevel perspective.

Analysis of time series data addresses the question on mechanisms underlying normal physiology and its alteration under pathological conditions. However, adding time variable to high-dimension, collinear, noisy data is a challenge in terms of mining and analysis. Here, we used Bayesian multilevel modeling for time series metabolomics in vivo study to model different levels of random effects occurring as a consequence of hierarchical data structure. A multilevel linear model assuming different treatment effects with double exponential prior, considering major sources of variability and robustness to outliers was proposed and tested in terms of performance. The treatment effect for each metabolite was close to zero suggesting small if any effect of cancer on metabolomics profile change. The average difference in 964 signals for all metabolites varied by a factor ranging from 0.8 to 1.25. The inter-rat variability (expressed as a coefficient of variation) ranged from 3-30% across all metabolites with median around 10%, whereas the inter-occasion variability ranged from 0-30% with a median around 5%. Approximately 36% of metabolites contained outlying data points. The complex correlation structure between metabolite signals was revealed. We conclude that kinetics of metabolites can be modeled using tools accepted in pharmacokinetics type of studies.

pH-Gradient Liquid Chromatography: Fundamentals and Examples.

In this paper, we acquaint the readers with the fundamentals of gradient separation, followed by the latest innovations in this field. We describe the principles of organic modifier- and pH-gradient elution emphasizing the differences and similarities with isocratic separation. The double organic modifier-/pH-gradient is also thoroughly reviewed as a useful method for the simultaneous determination of logkw (substitute of logP) and the pka of analytes present in complex mixtures.

Are Alpha-2D Adrenoceptor Subtypes Involved in Rat Mydriasis Evoked by New Imidazoline Derivatives: Marsanidine and 7-Methylmarsanidine?

The imidazoline compounds may produce mydriasis after systemic administration to some species (rats, cats, and mice). In mydriatic activity of imidazolines, α2D-adrenoceptors subtype(s) seems to be involved. In this study, the pupil dilatory effect evoked by 2 newly synthesized imidazoline derivatives-α2-adrenoceptor agonists: marsanidine and 7-methylmarsanidine-was compared. The compounds were tested alone as well as in the presence of α2-adrenoceptor antagonists (nonselective, yohimbine, and selective toward the following α2-adrenoceptor subtypes-α2A-2-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole maleate (BRL44408), α2B-2-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride (ARC239), α2C-JP1302, α2D-2-(2,3-dihydro-2-methoxy-1,4-benzodioxin-2-yl)-4,5-dihydro-1H-imidazole hydrochloride [RX821002]). The agonists were studied in male Wistar rats and were administered intravenously in cumulative doses. The antagonistic compounds were given in a single dose before the experiment with marsanidine or 7-methylmarsanidine. Pupil diameter was measured with stereoscopic microscope equipped in green light filter. Marsanidine and 7-methylmarsanidine exerted marked mydriatic effects. BRL44408, JP1302, and ARC239 did not cause significant parallel shift to the right of the dose-effect curves obtained for both imidazolines. In case of yohimbine and RX821002, the marked parallel shifts of dose-response curves were observed, with the antagonistic effects of RX821002 more pronounced. In vivo pharmacodynamics experiment suggests that α2D-adrenoceptor subtype is mainly engaged in mydriatic effects evoked in rats by imidazoline derivatives, in particular by clonidine.

Comparative pharmacodynamic analysis of imidazoline compounds using rat model of ocular mydriasis with a test of quantitative structure-activity relationships.

Imidazol(in)e derivatives, having the chemical structure similar to clonidine, exert diverse pharmacological activities connected with their interactions with alpha2-adrenergic receptors, e.g. hypotension, bradycardia, sedation as well as antinociceptive, anxiolytic, antiarrhythmic, muscle relaxant and mydriatic effects. The mechanism of pupillary dilation observed after systemic administration of imidazol(in)es to rats, mice and cats depends on the stimulation of postsynaptic alpha2-adrenoceptors within the brain. It was proved that the central nervous system (CNS)-localized I1-imidazoline receptors are not engaged in those effects. It appeared interesting to analyze the CNS-mediated pharmacodynamics of imidazole(in)e agents in terms of their chromatographic and calculation chemistry-derived parameters. In the present study a systematic determination and comparative pharmacometric analysis of mydriatic effects in rats were performed on a series of 20 imidazol(in)e agents, composed of the well-known drugs and of the substances used in experimental pharmacology. The eye pupil dilatory activities of the compounds were assessed in anesthetized Wistar rats according to the established Koss method. Among twenty imidazol(in)e derivatives studied, 18 produced diverse dose-dependent mydriatic effects. In the quantitative structure-activity relationships (QSAR) analysis, the pharmacological data (half maximum mydriatic effect - ED50 in µmol/kg) were considered along with the structural parameters of the agents from molecular modeling. The theoretically calculated lipophilicity parameters, CLOGP, of imidazol(in)es, as well as their lipophilicity parameters from HPLC, logkw, were also considered. The attempts to derive statistically significant QSAR equations for a full series of the agents under study were unsuccessful. However, for a subgroup of eight apparently structurally related imidazol(in)es a significant relationship between log(1/ED50) and logkw values was obtained. The lack of "predictive" QSAR for the whole series of the structurally diverse agents is probably due to a complex mechanism of the ligand-alpha2-adrenergic receptor interactions, which are predominantly of a highly structurally specific polar nature. Such interactions are difficult to quantify with the established chemical structural descriptors, contrary to the less specific, molecular bulkiness-related interactions.